This project utilizes an in vitro model of calcified cartilage differentiation to test the hypothesis that while the role of TGF-beta (TGF-B) in bone induction is to promote the initiation of chondrogenesis, subsequent development of a calcified cartilage phenotype requires the presence of additional osteoinductive factors in bone morphogenetic protein (BMP). The differentiation of mesenchymal cells to chondrocytes will be studied by incubating rat muscle mesenchymal cells with TGF-B alone or TGF-B with other BMP proteins. The cellular and molecular events involved in expression of the calcifying cartilage phenotype will be examined and compared with authentic resting zone and growth cartilage chondrocytes. Synthesis of cartilage specific proteoglycan and production of type II and type X collagen will be assessed using Northern and western blots and ELISA. Response to 1,25(0H)2D3 (growth cartilage cells) or 24,25(0H)2D3 (resting zone cells) will be assessed according to the differential activation of alkaline phosphatase (ALPase), cell number, and 3H-thymidine incorporation. Production of 1,25(0H)2D3 responsive, ALPase enriched matrix vesicles will be used as a marker for commitment to a calcifying cartilage phenotype. To determine whether response to TGF-B is transient or long lived, cultures and subclones will be reexamined following several passages and compared to cultures grown in agarose gels. Induced, resting zone, and growth cartilage chondrocytes will be incubated with BMP to determine whether further differentiation along a calcifying cartilage developmental cascade occurs. Whether BMP selects for subpopulations of TGF-B-induced chondrocytes will also be examined. Whether the induced chondrocyte populations can support matrix vesicle dependent mineralization will be determined and compared to calcification by growth plate chondrocytes.